Comrrriw \TTOUS
4.564
TO THE
Yol. 7 ' )
EDITOR
8.871, which was isomerized with alkali. The resulting iso-compound, without purification, was pyrolyzed giving a small yield of 4-chloro-7hydroxyphthalide, m.p. 155-15T0, which was identified by comparison with a known sample prepared by an unambiguous synthesis reported by Boothe, et al., in an accompanying communication.10
hydride in AT sodiuin hydroxide yielded 4-chloro-imethoxyphthalide-3-carboxylic acid (R = CHa, R' = H, R" = COOH) in 90y0yield; n1.p. 175176" with effervescence; 216 n i p ( 6 32,200); 240 mp ( E 8,240); 313 mp (E 5,220). ~ maX 0 .A 1J NaOH (after standing one hourj6 314 mp ( ~ 3 1 , 5 0 0 )285mp ; (€2,190). (10) J. H . Boothe, A. Green, J . P. Petisi, R G Wilkinson a n d C . \V Anal. Calcd. for C10H70QC1: C, 49.3; H, 2 . 9 ; Waller, THISJOURNAL, 79, 4664 (1957) C1, 14.6. Found: C, 49.5; H, 3.3; C1, 14.8. RESEARCH DIVISION 7 . S. \\'EBB The phthalidecarboxylic acid was then decnrORGANIC CHEMICAL R.W.~ R O S C H A R D RESEARCH SECTION D. €3. C O S G L I ~ Hboxylated by heating 5-10 minutes just above its AMERICAN cYAS.4MID COMPAXY LT. J. STEIN melting point to yield 4-chloro-7-methoxy-phthalPEARL RIVER,NEW YORK C. F.WOLF ide (R = CH3,R' = R" = HI which was sublimed RECEIVED AIJCL-ST 1, 195'7 at 175" (760 mm.); yield, 705%; m.p. 167-168" ~ 0 . S1 H C l 215 < nip (. 37,300); 236 nip ( E S,S30); mp ( E 4,560); A~:;vNaoH (after standing one DEMETHYLTETRACYCLINES. SYNTHESIS OF A DEGhour)6286 mp (E 2,190). RADATION PRODUCT Sir: Anal. Calcd. for C9H7OaC1: C, 54.4; 13, 3 . 6 : In an accompanying communication' there is C1, 17.9. Found: C, 54.8; H, 3.7; C1, 17.7. described a new series of antibiotics closely related The methyl ether was cleaved by refluxing in to the tetracyclines, both in antibacterial activity 48y0hydrobromic acid for 2.5 hours. The product, and in structure. In a second accompanying 4-chloro-7-hydroxyphthalide (R = K' = K" = H ) , communication* evidence has been presented that crystallized from the hydrobromic acid on coolirigthese new antibiotics differ from the parent corn- in 70:%, yield and was then sublimed at 100"(15.-20 pounds only in that they lack a methyl group at rnm.). The m.p. was 158-159" and there was no the 6-position of the tetracycline nucleus. depression upon admixture with the degradation Ilye now wish to report the synthesis of a deg- product.' The ultraviolet and infrared spectra were radation product which proves that the arrange- identical; A~:;'""' 235 mp ( E 8,400); 308 nip O . 1 S s a O H-54 3 mp ( E 5,400); 343 111p ment of substituents in the D ring and in portions ( e 4,]*50);~ "nx of the C ring of demethylchlorotetracycline is the ( E 6,180). same as in chlorotetracycline except for the C-6 Anal. Calcd. for C8H6O3C1: C, 52.1; H, 3.7; methyl group. This conipound is 4-chloro-7C1, 10.2. Found: C, 52.2; H, 3 . 2 ; C1, 19.0. hydroxyphthalide (R = R ' = R" = H) which is analogous to 4-chloro-7-hydroxy-3-rnethylphthalide (6) Upon standiug in 0.1 N sodium hydroxide f o r a n hour or less the long wave length absorption maximum undergoes a hypsochrolnic (R = R' = H , R" = CH3), obtained by the same shift which is reversible b y acidification. This is assumed t o be tlegradative route from chlorotetracycline. attributable t o t h e opening and closing of t h e lactone ring and will
37;
R'
c1
A,/"'
0:) OR
O \
be dealt with in mare detail in a subsequent publication.
RESEARCH DIVISIOX
ORGAXIC CHEMICAL
J. H. BOOTHE A . GREEX
J. P. PETISI R. G. WILKIXSON PFARI.RIVER,NEWYORK C . W.\VAI.I.ER RECEIVED AUGUST1, 1967
RESE.4RCH S E C T I O S
h E R 1 C A X CYAN.4MID COMP-4NY
The starting point for the synthesis is 4-chloro-3or,or'-DIGLYCEROPHOSPHATEI N PLANTS = R' = CH3, K" = OH) whose synthesis has already been Sir: reported from this laboratory. This compound We have observed a P3*-labeled compound in was oxidized with potassium permanganate in 0.5 hydrolysates of Scendesmus phosphatides which N sodium hydroxide a t 90" for one hour to contained more than a third of the lipid phosphorus. yield the 4-chloro-3-hydroxy-7-methoxyphthalideThe same phosphate ester also possessed as much 3-carboxylic acid (R = CHa, R' = OH, R" = as 90% of the alcohol-soluble non-lipid phosphorus COOH) in 67y0 yield. This compound has been of Scenedesmus cultured at low light intensity in isolated as a degradation product of chlorotetra- media containing P32. The cellular concentration cycline and was named there as the tautomeric of the ester, calculated from its Pa2activity and thcl acid.5 The nutrient specific activity, was as high as 10-3 -1r. keto-acid, 6-chloro-3-methoxyphthalonic reduction of this compound with sodium boro- The same compound in lower concentrations oc(11 J. R. D. McCormick, N. 0. Sjolander, U. Hirsch, E. R . Jensen curred in the only two species of higher plants 79, 4561 (1957). and A. P. Doerschuk, THISJOURNAL, (clover) tested. I t was isolated by chromatog( 2 ) J , S. Webb, R . W. Broschard, D. B. Cosulich, W. J . Stein, and raphy on Whatman KO. 1 paper with Rf = 0.36 C . F. Wolf, ibid., 79, 4563 11957). in phenol-water and Rf = 0.11 in butanol-pro(3) C . R . Stephens, L. H . Conover, R . Pasternack, F. A . Hochstein, W. T. hloreland, P. P . Regna, F. J . Pilgrim, K. J . Brunings and pionic acid-water.' These RE values correspond R . B . Woodward, { b i d . , 76, 3568 (19.54). to those recorded by Dawson2 for an unknown i i i (4) J. H. Boothe, S. Kushner, J. P. Petisi and J. H. Williams, i b i d . , rat liver extracts. 75, 3261 (1953).
hydroxy-7-methoxy-3-methylphthalide (R
( 5 ) B. L. Hutchings, C . W. Waller, S. Gordon, R. W, Broschard, C. F. Wolf. A. A. Goldman and J. H . Williams, ibid., 74, 3710 11952). For a discussion of and references to this t y p e o f t a u t ~ m e r i s msee ref. 4.
(1) A A. Benson, J. A Bassham, M. Calvin, T C G o o d d e , V A Haas and W Stepka. TRISJ O U R N A L , 73, 1710 (1950) 12) R h I C Dawson, Biuchim el B t o p k y s Acto, 14, 374 f l Y 5 4 )
.\ug 20 19.77
COMSITJNICATIONS TO TIIE EDITOR
4,565
The ester was half hydrolyzed during two min- havior suggested to us the possibility that aliphatic utes a t 100” in 1.0 N nitric acid to give only one compounds of type 11 might be resolvable into reaP32-labeled product which was identified as glyc- sonably stable optical antipodes a t room temperaerophosphate by paper chromatography and elec- ture since the barriers for rotation about the centrophoresis. The unknown was anionic in the pH RI Rn R: range 2-12 and readily separable from the known F R3 1 1 IIa Br F Br glycerophosphoryl esters of the phospholipids. R,-C-C-R? IIb Br Br C1 Scenedesmi~s-C~~ was cultured and the unknown ITc Rr C B H ~ Br 1 1 was isolated and identified with the P32-labeled F RB compound. Acid hydrolysis produced equal acI1 tivities of glycerophosphate and glycerol which was tral C-C bond of TI a-c should be substantially identified by its R,+ values and by quantitative periodate oxidation to formaldehyde and formate. greater than for Ia-d. This expectation was not realized. The nuPeriodate oxidation of the ester indicated an a,a’structure. Synthetic a ,a’-diglycer~phosphate~ was clear magnetic resonance spectra of IIa-c showed prepared and found inseparable from the radio- equivalent gem-fluorines down to - 30”. Thus active unknown by paper chromatography and IIa shows doublet CF2 and triplet C F absorptions electrophoresis. Its identical hydrolysis rate was separated by 440 C.P.S. with J equal to 15-19 C.P.S. determined by neutron activation Chromatography The results indicate that there is rapid rotation at of the products. room temperature about the central C-C bond in Tetraacetyal bis- (L-a-glyceryl) phosphates have IIa-c and, by inference, in Ia-d also.3 The solution to the apparent paradox is that the been synthesized by B ~ i e r . ~Polyglycerophosphate shift between gem-groups as in compounds structures in several lipids have been r e p o r t e d j ~ ~ .chemical ~ but diglycerophosphate has not been identified pre- like I is not necessarily averaged by rapid rotation viously. Its symmetry, simplicity and acid lability unless the residence times of the molecule in each of the to give the usual products of lipid hydrolysis ap- various rotational conformations ( I I I ) are equal. parently have postponed its identification as a The gem-group does not have equivalent atoms in metabolite. Its occurrence in the glyceryl phos- any of the configurations IIIa, b or c. Therefore phatides will be reported. IVe are indebted to Dr. R. J. Suhadolnik for invaluable assistance in the early stages of the identification. This work was supported by the National Science Foundation, the Atomic Energy Commission and the Pennsylvania Agricultural Experiment Station. ( 3 ) E. Fischer and E. Pfahler, B e y . , 63, l G O G (1920). (4) E. Baer, J. Biol. Chem., 198, 853 (1962). ( 5 ) M. C. Pangborn, ibid., 168, 361 (1947). (6) J. M. McKibbin and W. E. Taylor, ibid., 198, 853 (1952). (7) P. Fleury, Bull. SOC. Chim. B i d , 30, 521 (1948).
DEPARTMEST O F AGRICULTURAL A S D BIOLOGICAL CHEXISTRY PENNSYLVANIA STATEUNIVERSITY USIVERSITYPARK, PENSSYLVANIA RECEIVED JULY 3, 1957
B. MARUO A . A. BENSON
NUCLEAR MAGNETIC RESONANCE SPECTRA. HINDERED ROTATION AND MOLECULAR ASYMMETRY’
Sit,: The elegant and unequivocal demonstration of non-equivalent fluorine atoms in certain gemdifluoro compounds of type I with the aid of their nuclear magnetic resonance spectra has been interpreted as indicating restricted rotation about the central C-C bond of I.?
unless the residence times in each configuration are equal, the chemical shift difference between the gem-fluorines will not average out in the general case.4 Since the relative residence times will be temperature dependent, the apparent degree of non-equivalence of the fluorines will vary with temperature as is observed.? RI Rz XI Ra With more symmetrical compounds like 11, F R9 rapid rotation a t > -30” will average the chemical Ia Br H C1 R r Ishifts of the gem-difluorines since the residence R ,-C-C--R? Ib Br F C1 Br times (and populations) for the two configurations IC Rr H Br CBHS I 1 I F Rd Id C1 €1 C1 CBHS with non-equivalent fluorines (IVb,c) must be (3) Drs. W. D Phillips and J. J. Drysdale have informed us t h a t Significantly, spectra for the “freely rotating” substances with “equivalent” gem-fluorines were not they have independently made similar observations on fluorine comof related structure. produced a t temperatures up to 200”.2 This be- pounds (4) I n some situations, t h e spin-spin coupling between the non-
’
(1) Supported in part by t h e Office of Naval Research. J J Drysdale and XV D Phillips, THISJ O U R N A L , 19, 310 (1957). (2)
equivalent fluorines, or other similarly located atoms, may be so large a s to swamp o u t t h e chemical shift and give t o all intents and purposes n single resonance line.
